Figure 3. Gephyrin is not SUMO1-conjugated in vivo and in vitro.
(A) SDS-PAGE (4–12%) followed by anti-gephyrin Western blot analysis of input and HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His6-HA-SUMO1 KI brains. The presence of gephyrin in both WT and KI eluates indicates non-specific binding of gephyrin to the affinity matrix. (B) Representative SDS-PAGE (10%) followed by Western blot analysis of input and eluate fractions of anti-gephyrin and anti-IgG immunopurifications in the presence of 20 mM NEM from WT and His6-HA-SUMO1 KI brains. Anti-gepyhrin Western blot confirms the enrichment of gepyhrin in both WT and KI brains after affinity purification using anti-gephyrin antibody but not when using mouse IgG (black arrow, upper panel). Importantly, anti-HA Western blot analysis does not reveal SUMO1-gephyrin bands. (C) SDS-PAGE (10%) followed by Western blot analysis of input and eluate fractions of anti-HA immunoprecipitation in the presence of 20 mM NEM from HEK cells overexpressing HA-SUMO1 and GFP-gephyrin, alone or in combination. Anti-HA Western blot analysis confirms the enrichment of HA-SUMO1 conjugates (lower panel) but no SUMO1-gephyrin signal is observed in the eluate fractions (upper panel). Images are representatives of at least three independent experiments.